Metabolic Biochemistry / BIBC 102 Midterm Exam / Spring 2005 I. (20 points) Fill in all of the enzyme catalyzed reactions which convert glycogen to lactate. Draw the correct structure for each intermediate molecule, showing the correct position of the hydrogen atoms at each carbon atom. Include the names or abbreviations of any other reactants and products (eg, ATP, ADP, Pi, NAD+, NADH, etc), and any required prosthetic groups (eg, biotin, TPP, etc.). (Note:You do not need to give the correct name for each intermediate molecule or the correct name of the enzyme to receive full credit. Partial credit will, however, be given for the correct name of an intermediate structure if the structure itself is incorrect.) Glycogen (draw the 2 terminal sugar subunits of the polymer only) lactate (draw structure)
2 II. (20 points) A. Given a mitochondrial extract with all of the enzymes and cofactors normally found inside mitochondria but lacking pyruvate dehydrogenase completely, show how pyruvate, CO2, and acetyl CoA can be used to form succinate when succinate dehydrogenase is inhibited by malonate. (Assume there is no oxaloacetate initially present.) Draw the correct structure for each intermediate molecule, showing the correct position of the hydrogen atoms at each carbon atom. Include the names or abbreviations of any other reactants and products (eg, ATP, ADP, Pi, NAD+, NADH, etc), and any required prosthetic groups (eg, biotin, TPP, lipoate, etc.). B. Show the path of 14 C label in each molecule above starting with 14 C label in the methyl carbon of pyruvate ( 14 CH 3 -C(O)-CO 2 -).
3 III. (25 points) (a) Draw a diagram of the overview of proton and electron circuits in mitochondria starting with NADH in the matrix and ADP and P i in the cytosol and ending with the reduction of oxygen to water and the formation of ATP (the diagram in the lecture handout will receive full credit). Label each complex and protein, identify matrix, intermembrane space, and inner membrane, and indicate the flow of protons and electrons. (b) Indicate on this diagram the relevant electron carriers in each complex of electron transport (e.g., FeS, cytochrome a 3, Cu, FMN, etc.).
IV. (15 points) (A) In the space below please sketch the v versus [S] plot for an enzyme obeying Michaelis- Menten kinetics in the presence and absence of a competitive inhibitor. Assume an inhibitor concentration of [I] = Ki. Please label lines, and indicate Vmax and Km for each plot. 4 (B) In the space below, please sketch the v versus S plot for an enzyme which shows positive cooperativity in substrate binding, in the presence and absence of an allosteric inhibitor. What form of the allosteric enzyme binds the inhibitor better, the R state or the T state? (C) The graph below shows the v versus [S] plot for phosphofructose kinase and its substrate ATP. Explain the shape of this curve. (Hint: consider the different sites on PFK to which ATP can bind).
5 VI. (20 points) Acceptor control. In the following problem, you are given purified, intact mitochondria, an abundant source of matrix NADH, O 2, and phosphate. You are also given the following diagram: A. Please draw the lines expected for the time course of O 2 consumption following the addition of 50 and 100µmoles of ADP (you should plot the results of the two separate experiments as two lines emanating from point a on the graph). (Assume that there are 30µmoles of O 2 initially present). B. Does the rate of O 2 consumption differ in the two experiments? If so, by what factor does it differ? C. What is the final number of ATP molecules produced from 50µmoles of ADP at the point that further O 2 consumption ceases? How many O 2 molecules have been consumed at this point? For full credit, please show your calculations. D. Why is the rate of O 2 consumption essentially zero prior to the addition of ADP?
VI. (100 points) Multiple Choice. Choose only one answer for each question. 6 1. Which one of the following statements is blatantly false? (A) In a parallel β pleated sheet structure, all of the R groups project to the same side of the β sheet plane, and so define the character of this surface of the sheet. (B) The amino acids R, D, and glutamine are all likely to be found on the exterior of a globular protein. (C) The amino acids L, V, and isoleucine are all likely to be found in the interior of a globular protein. (D) In an α helix, all of the R groups project outward from the helix core and define the character of the α helix surface. (E) Secondary structure refers to the ordered, hydrogen-bonded structures of a protein (e.g., α helix and β pleated sheet.) 2. Which one of the following statements is blatantly false? (A) Km, the Michaelis constant, is the substrate concentration at which the reaction velocity, v, is half of Vmax, and the units of Km are therefore molar. (B) The velocity of an enzyme catalyzed reaction, v, is proportional to the enzyme concentration, [E], when [S] >> Km, but not when [S] << Km. (C) Enzymes decrease G, the activation free energy, for the reactions they catalyze by selectively stabilizing the transition state. (D) Competitive inhibitors bind reversibly to the active site of an enzyme and prevent binding of the substrate. (E) An enzyme obeying Michaelis Menten kinetics will achieve 2/3 of Vmax when [S] = 2 Km. 3. Which one of the following statements is blatantly false? (A) Positive cooperativity in substrate binding is never observed for enzymes that have only one active site. (B) When an allosteric inhibitor binds to the allosteric site on an enzyme, it shifts the R/T equilibrium to the T state. (C) The phosphorylation of a specific serine residue in each subunit of glycogen phosphorylase activates the enzyme by shifting the R/T equilibrium to the R state. (D) Irreversible enzyme inhibitors typically inactivate enzymes by forming covalent bonds with an active site amino acid. (E) For an allosteric enzyme, the substrate concentration at which half maximal velocity is attained, called K 0.5, will be greater if an allosteric activator is present. 4. The synthesis of the activated form of acetyl CoA (acetyl-coa) is carried out in an ATP-dependent process catalyzed by acetyl CoA synthase: Acetate + CoA + ATP acetyl CoA + AMP + PPi The pyrophosphate product (PPi) of this reaction is rapidly hydrolyzed to 2 Pi in a reaction carried out by pyrophosphatase. What is the G for this reaction when the final product is 2Pi (in kj/mol)? You are given: G for hydrolysis of acetyl CoA to acetate + CoA is -32.2 kj/mol; G for hydrolysis of ATP to AMP + PPi is -30.5 kj/mol; and G for hydrolysis of PPi to 2 Pi is -33.4 kj/mol. (A) -31.7 (B)+1.7 (C) +31.7 (D) +63.9 (E) -96.1 5. Coupling ATP cleavage to an unfavorable reaction. Consider a hypothetical biosynthetic reaction A B whose equilibrium constant K = [B] / [A] = 10-4. In vivo, this reaction is coupled to the hydrolysis of ATP: A + ATP B + ADP + Pi, whose equilibrium constant is 10 2. By what factor does coupling the formation of B to the hydrolysis of ATP increase the ratio of [B] to [A] when the phosphorylation potential in the cell ( [ATP] / [ADP][Pi]) is 10 3? (A) 10 7 (B) 10 8 (C) 2 x 10 8 (D) 5 x 10 8 (E) 10 9
7 6. Which one of the following statements is blatantly false? (A) At steady state in a typical cell, the reactions catalyzed by aldolase and enolase are essentially at equilibrium. (B) Adenylate kinase catalyzes the reaction: AMP + ATP 2 ADP (C) An increase in the phosphorylation potential (i.e., the [ATP]/[ADP][Pi] ratio) will increase the rate of a biosynthetic pathway. (D) For the phosphofructokinase reaction in a typical cell, the ratio of the products (fructose-1, 6- bisphosphate and ADP; numerator) to the reactants (fructose-6-phosphate and ATP; denominator) to is larger than if the reaction were at equilibrium. (E) Under steady state conditions in a typical cell, the ability of ATP hydrolysis to do useful work (e.g., drive biosynthetic reactions) is given by the actual free energy change, G, a value that is substantially more negative than the standard free energy of ATP hydrolysis, G = -30.5 kj/mol. 7. Which one of the following statements is blatantly false? (A) The net ATP equivalents produced per glucose subunit of glycogen in an anaerobic muscle cell is 3. (B) The net ATP equivalents produced from glucose in an anaerobic muscle cell treated with arsenate is 0. (C) If glucose is labeled at carbon 4, pyruvate will be labeled at the carboxyl carbon. (D) If glucose is labeled at carbon 1, ethanol will be labeled in the methyl carbon. (E) In a yeast cell that lacks triose phosphate isomerase, none of the pyruvate formed from glucose-6-14 C will be labeled. 8. Which one of the following statements is blatantly false? (A) Glycogenolysis and Glycolysis occur in the cytosol, not in mitochondria (B) Lactate dehydrogenase is found in the cytosol, not the mitochondria (C) Pyruvate dehydrogenase and the citric acid cycle are found in mitochondria, not in the cytosol. (D) The non-oxidative branch of the pentose phosphate pathway (conversion of ribose-5-phosphate to glycolysis intermediates ) occurs in the cytosol, not in mitochondria (E) The oxidative branch of the pentose phosphate pathway (conversion of glucose-6-phosphate to ribose-5- phosphate) occurs in mitochondria, not in the cytosol. 9. Which one of the following statements is blatantly false? (A) Pyruvate dehydrogenase kinase is allosterically inhibited by NADH. (B) Glycogen phosphorylase b is allosterically activated by AMP. (C) Phosphofructokinase is allosterically activated by AMP. (D) Pyruvate kinase is allosterically inhibited by ATP. (E) Pyruvate carboxylase is allosterically activated by acetyl CoA. 10. You are studying catabolism and electron transport in a minced tissue preparation of pigeon breast muscle which contains all of the enzymes of glycolysis and intact mitochondria. You already know that this tissue is capable of perfectly normal aerobic and anerobic glycolysis. Furthermore the TCA cycle and electron transport can all function normally. Now in the presence of O 2 and glucose, you add an inhibitor of electron transport to the aerobically respiring tissue. Which one of the following possible results are you least likely to see? (A) O 2 consumption falls dramatically. (B) The NADH/NAD ratio in the mitochondria increases. (C) All CO 2 production from glucose ceases. (D) All ATP synthesis from glucose ceases. (E) The rate of glucose consumption increases
8 11. When O 2 is added to a suspension of yeast cells which have been growing anaerobically on glucose, the rate of glucose consumption falls dramatically and ethanol ceases to be produced. Which one of the following statements about this effect, called the Pasteur effect, is blatantly false? (A) The concentration of NADH in the mitochondrial matrix will decrease when O 2 is added. (B) The phosphofructokinase reaction will no longer be activated by high AMP levels when O 2 is added. (C) Assuming ATP needs are constant, the theoretical decrease in glucose consumption when O 2 is added is 15-fold. (D) Assuming ATP needs are constant, the rate of CO 2 evolution will increase when O 2 is added. (E) The ratio of [ATP]/[ADP][P i ] will increase when O 2 is added. 12. Which one of the following statements is blatantly false? (A) The substrate for the first enzymatic reaction in the pentose phosphate pathway is glucose-6-p. (B) Under aerobic conditions, the fate of electron pairs in NADH is to reduce O2 to H2O with the production of ATP, while the fate of electron pairs in NADPH is to reduce bonds in biosynthetic reactions. (C) The steady state NADPH/NADP + ratio is far larger than the steady state NADH/NAD + ratio in cells. (D) There are two dehydrogenase reactions in the oxidative branch of the pentose phosphate pathway, both of which are absolutely specific for NADP+. (E) The prosthetic group of transaldolase, an enzyme found in the non-oxidative branch of the pentose phosphate pathway, is thiamine pyrophosphate. 13. Which one of the following statements is blatantly false? (A) Thiamine pyrophosphate is a prosthetic group of the E1 subunit of pyruvate dehydrogenase. (B) If tritium (T) is attached to the middle carbon of lactate, lactate dehydrogenase will catalyze the transfer of this tritium atom plus an electron pair to the nicotinimide ring of NAD+ to form NADT. (C) The prosthetic group for E 3 subunit of pyruvate dehydrogenase is FMN. (D) In the pyruvate carboxylase reaction, biotin is first carboxylated by CO 2 at one active site in a reaction which is driven by the coupled hydrolysis of ATP and then, at the other active site, the carboxyl group of carboxybiotin is transferred to pyruvate. (E) The order in which substrates are used in the pyruvate dehydrogenase mechanism is pyruvate, then CoA- SH, then NAD +. 14. Which one of the following statements is blatantly false? (A) If intact mitochondria are incubated with O 2, ADP, Pi, and a source of matrix NADH, the addition of antimycin A will cause ubiquinone to become fully reduced and cytochrome c to become fully oxidized. (B) If intact mitochondria are incubated with O 2, Pi, and a source of matrix NADH, the addition of ADP would be expected to decrease the NADH/ NAD ratio. (C) If 14 C labeled NADH is added to a suspension of intact mitochondria in the presence of oxygen, ADP, and P i, 14 C radioactivity will appear in the mitochondrial matrix. (D) The exergonic transfer of two electrons from NADH to UQ using complex I provides the energy to pump 4 H + out of the mitochondria. (E) The respiratory quotient (ratio of CO 2 generated/o 2 consumed) for glucose is 1. 15. Which one of the following statements is blatantly false? (A) In the glycerol phosphate shuttle, the inner mitochondrial membrane glycerol phosphate dehydrogenase transfers electrons from its FADH 2 prosthetic group to Complex I. (B) In intact mitochondria, electron transport will cease when the energy it takes for electron transport to pump a proton against a concentration gradient equals the energy obtained from the exergonic transfer of the electron pair from NADH to oxygen. (C) In the glycerol phosphate shuttle, the substrates for the cytosolic enzyme glycerol phosphate dehydrogenase are NADH and dihydroxyacetone phosphate. (D) Cyanide inhibits electron transport by binding to the Fe 3+ form of cytochrome a 3. (E) Electron transport complex II is also known as succinate dehydrogenase.
16. Which one of the following statements is blatantly false? (A) Oligomycin binds to ATP synthase and thereby inhibits ATP formation. (B) Atractyloside binds to the adenine nucleotide translocase, and prevents the transport of ADP into the mitochondria, and the transport of ATP out of the mitochondria. (C) Rotenone inhibits electron transport from Complex II to ubiquinone. (D) All cytochromes consist of the heme prosthetic groups bound to a protein. (E) Cytochrome oxidase (also called Complex IV) accepts electrons one at a time from cytochrome c and then transfers 4 electrons to O2 in order to form 2 H2O without generating oxygen radicals or peroxides. 17. A muscle cell is actively engaged in the complete aerobic catabolism of glucose to 6 CO 2. What will be the net yield of ATP equivalents (i.e., ATP, GTP, UTP, etc.) from each glucose when excess DNP is present? (A) 2 (B) 4 (C) 6 (D) 30 (E) No ATP can be produced 18. When a cell extract containing all of the enzymes of glycolysis and the TCA cycle is incubated aerobically with Glucose-2-14 C, what percentage of the original label will remain in oxaloacetate after two turns of the TCA cycle? (In this problem assume that any label which enters the cycle does so in the first citrate synthase step, and that the acetyl CoA entering in the second and later turns of the TCA cycle is unlabeled.) (A) 0% (B) 25% (C) 50% (D) 75% (E) 100% 19. A cell extract containing all of the enzymes of glycolysis is incubated with 32 P labeled ATP (i.e., Adenosine - P - P - 32 P) and glucose. Assuming that inorganic phosphate is unlabeled, which of the following labeled compounds would be formed? (1) Glucose-6-32 P (3) Fructose-1-32 P, 6-32 P-bisphosphate (2) 2-32 P phosphoenol pyruvate (4) 1 P, 3-32 P-bisphosphoglycerate (A) 1 and 3 (B) 1, 2, and 3 (C) 2 and 4 (D) 4 (E) All of the above 20. The following graph represents the hypothetical results of an experiment in which the amount of O 2 consumed is measured over a time interval during which intact mitochondria are treated sequentially with five substances. The identity of these 5 substances can be inferred from their effect on the amount of O 2 consumed. Assume that P i and an abundant source of matrix NADH are present. 9 (A) 1 = ADP 2 = Oligomycin 3 = DNP 4 = antimycin A 5 = Succinate (B) 1 = ADP 2 = Oligomycin 3 = Arsenate 4 = antimycin A 5 = Succinate (C) 1 = ADP 2 = Oligomycin 3 = DNP 4 = Rotenone 5 = Glycerol P (D) 1 = DNP 2 = atractyloside 3 = succinate 4 = antimycin A 5 = Ascorbate (E) 1 = DNP 2 = Atractyloside 3 = ADP 4 = cyanide 5 = succinate
21. You will find below several correctly drawn structures of important compounds in biochemistry. Which of the following are represented? (1) NADPH (2) FMNH 2 (3) UQH 2 (4) lipoic acid (5) Lysine (A) 1,3,5 (B) 1,3,4 (C) 1,2,3 (D) 1 and 5 (E) 2 and 4 10